Biological interactions at the molecular level are controlled in part by how tightly various molecules bind one another. Binding between molecules may be used to characterize, select, separate and/or isolate molecules. The present techniques for characterizing, selecting, separating and/or isolating molecules based upon binding, however, are not ideal for use in connection with many molecules.
One measure of the binding between molecules is "affinity". Affinity may be described as the tightness of binding between two molecules at equilibrium. Another measure of binding is the "on-rate", which describes the relative rate at which two unbound molecules tend to form a complex. "Off-rate" describes the relative rate at which a complex of two molecules tends to dissociate.
Affinity separations typically have been carried out using a column with the target molecule ("acceptor") covalently coupled to a solid phase within the column. The test molecule ("ligand") then may be poured over and gravity fed through the column. Ligands having a high affinity for the bound acceptors and low off-rates then will bind to the column with all other molecules passing through the column. The bound ligand molecules then may be eluted from the column using an elution buffer.
This procedure, while acceptable for ligands with high affinities and low off-rates, has many drawbacks. Large amounts of acceptors are required to prepare the column. The acceptor also must be covalently attached to the solid support which typically involves harsh conditions potentially affecting the structure of the acceptor and the affinity between the acceptor and ligand. Moreover, not all acceptors are easily susceptible to covalent linkage to an appropriate solid phase. The procedure also cannot be used when affinity is low and/or off-rate is high. Furthermore, although the procedure can be useful to determine whether two molecules bind tightly, it does not readily yield information on how tight the binding is, or it reveals that information in a manner that cannot be used to determine the "affinity constant" (the affinity constant being the generally accepted numerical representation of "tightness" of binding at equilibrium).
One procedure not requiring covalent linkage of the acceptor to the solid phase of a column has been described. This procedure involves fractionating the ligand on two gravity-fed, gel-filtration columns In one column no acceptor molecules are present. In the other column, acceptor molecules are present throughout the column and in all the liquid that will be passed through the column. The rate at which the ligand moves down each column is a measure of its size. Consequently, if the ligand binds the acceptor in the second column it will move at a rate appropriate for a molecule of larger size.
The procedure has many drawbacks. Large amounts of acceptor are required. The procedure also lacks the sensitivity to detect small changes in molecular size, because nonuniform flow through the column limits resolution. The procedure further depends on preparing two identical columns or on running the same column twice under exactly identical conditions, which can be very difficult
An affinity electrophoresis technique involving antibodies or antigens coupled to a gel has been described. According to this procedure, antibodies or antigens are dispersed throughout and covalently linked to the solid phase of a gel. The test molecules then are electrophoresed through the gel containing the immobilized molecules. This procedure has among its drawbacks the problems created by covalent coupling to a solid phase.